Use of calmodulin kinase II inhibitors to treat myocardial dysfunction in structural heart disease

ABSTRACT

The present invention provides a method for treating structural heart disease in a subject, comprising administering an effective amount of an inhibitor of CaMKII to the subject, whereby the administration of the inhibitor treats the structural heart disease in the subject. Also provided are transgenic animal models for treating structural heart disease. Further provided is a means of screening for a compound that can treat structural heart disease.

[0001] This application claims priority to U.S. provisional applicationsSer. No. 60/326,576 filed Oct. 1, 2001, and Ser. No. 60/328,010, filedOct. 8, 2001. The 60/326,576 and the 60/328,010 provisional patentapplications are herein incorporated by this reference in theirentirety.

[0002] This invention was made with government support under HL03727 andHL62494 awarded by the National Institutes of Health. The government hascertain rights in the invention.

BACKGROUND

[0003] 1. Field of the Invention

[0004] The invention relates to the inhibition of Calmodulin kinase II(CaMKII). More specifically, the CaMKII inhibition can treat or preventstructural heart disease, for example contractile dysfunction followinga myocardial infarction, or dilated cardiomyopathy.

[0005] 2. Background Art

[0006] Myocardial infarction is a major cause of significant disabilityand death in the United States and in many other countries around theworld, and accounts for approximately {fraction (2/3)} of all heartfailure.⁷

[0007] Several disease-initiating events (e.g. myocardial infarction,untreated hypertension, congenital mutations of contractile proteins)can result in a common heart disease phenotype that consists of dilationof the cardiac chambers, resulting in reduction in contractile function(i.e., a decrease in the fraction of total blood ejected from eachchamber during systole) that leads to the clinical syndrome of heartfailure.⁷ Dilated cardiomyopathy includes two distinct disease entities.Dilated cardiomyopathy, as used herein, includes ischemic cardiomyopathywhich is a disease entity characterized by left ventricular dilation andreduced contractile function. This condition can result after myocardialinfarction when the normal compensatory hypertrophy of surviving,non-infarcted myocardium is insufficient.⁷ “Dilated cardiomyopathy” canalso include increased myocardial mass and reduced contractile functiondue to a genetic abnormality of myocardial proteins in the absence ofmyocardial infarction.⁷ A subject with dilated cardiomyopathy is asubject who has decreased cardiac contractility due to dilation of theventricles. Thus, a subject with dilated cardiomyopathy and contractiledysfunction has different and more severe dysfunction than a subject inwhom hypertrophy of surviving non-infarcted myocardium has compensatedfor infarcted myocardium. Further, a subject with dilated cardiomyopathyand contractile dysfunction has disease that is distinct from othercardiac conditions including abnormal relaxation (i.e., diastolicdysfunction and cardiac arrhythmia.

[0008] Available therapies for heart failure are insufficient, and newmethods of treatment are needed. The heart responds to infarction byhypertrophy of surviving cardiac muscle in an attempt to maintain normalcontraction. However, when the hypertrophy is insufficient tocompensate, dilated cardiomyopathy and reduced contractile functionresult, leading to heart failure and death.¹⁹ Despite important advancesin medical therapies for preventing cardiac dysfunction and heartfailure after myocardial infarction,¹⁵ these problems remain asignificant unsolved public health problem.

[0009] No pharmacological therapy for dilated cardiomyopathy is curativeor satisfactory, and many patients die or, in selected cases, undergoheart transplantation. Presently available pharmacological therapies forreducing cardiac dysfunction and reducing mortality in patients withheart failure fall into three main categories: angiotensin-convertingenzyme (ACE) inhibitors, beta adrenergic receptor (OAR) antagonists, andaldosterone antagonists. Despite reducing mortality, patients treatedwith these medicines remain at significantly increased risk for deathcompared to age-matched control patients without heart failure. ACEinhibitors,¹¹βAR antagonists⁴ and (at least one type of) aldosteronereceptor antagonist¹²can significantly reduce the incidence and extentof cardiac dysfunction and heart failure after myocardial infarction.Other available pharmacological therapies include nitroglycerin,diuretics, positive inotropic agents (cardiac stimulants), and brainnatriuretic peptide (BNP). These latter agents can provide symptomaticrelief, but are not associated with reduced mortality in heart failurepatients.

[0010] ACE inhibitors are associated with cough in 10% of patients andcan result in renal failure in the setting of bilateral renal arterystenosis or other severe kidney disease.⁷βAR antagonists are associatedwith impotence and depression, and are contraindicated in patients withasthma; furthermore, patients may develop worsened heart failure,hypotension, bradycardia, heart block, and fatigue with initiation ofβAR antagonists⁷ Aldosterone receptor antagonism causes significanthyperkalemia and painful gynecomastia in 10% of malepatients.^(7,12)Agents without a demonstrated mortality benefit are alsoassociated with problems; most notable is the consistent finding thatmany cardiac stimulants improve symptoms, but actually increasemortality,⁷ likely by triggering lethal cardiac arrhythmias. Incontrast, CaMKII inhibition is now known to reduce cardiac arrhythmiasin animal models,^(20,21)and so represents a novel approach to enhancingcardiac function without increasing arrhythmias. Presently availablepharmacological therapies are ineffective and are limited by significantunwanted side effects, and so development of new therapies with improvedefficacy and less severe side effects is an important public healthgoal.

[0011] Calmodulin kinase II is an enzyme that is present in heart and isactivated when Ca²⁺ increases inside the heart cells, and binds to theCa²⁺ binding protein calmodulin.³ CaMKII activity can increase inpatients with severe cardiomyopathy, but CaMKII has never been linked todilated cardiomyopathy or deterioration of contraction in heart failure.

[0012] The present invention provides methods of improving (increasing)contractile function of the myocardium to treat dilated cardiomyopathyand heart failure by inhibiting CaMKII. The present invention furtherprovides mouse models of cardiac-targeted CaMKII inhibition bytransgenic over expression of a selective CaMKII inhibitory peptide,AC3-I. Thus, the present AC3-I transgenic mouse is an important new toolto test for the effects of chronic CaMKII inhibition in cardiac disease.

SUMMARY OF THE INVENTION

[0013] The present invention provides a method of treating or preventingmyocardial dysfunction that follows myocardial infarction in a subject,comprising administering to the subject an effective amount of aninhibitor of Calmodulin Kinase II (CaMKII), whereby the administrationof the inhibitor improves myocardial contraction after a myocardialinfarction in the subject.

[0014] The present invention provides a method of treating or preventingmyocardial dysfunction that occurs in dilated cardiomyopathy or otherstructural heart disease (e.g., end-stage valve disease) in a subjectdiagnosed with dilated cardiomyopathy or other structural heart disease,comprising administering to the subject an effective amount of aninhibitor of CaMKII, whereby the administration of the inhibitor treatsor prevents dilated cardiomyopathy or other structural heart disease inthe subject.

[0015] The present invention provides a method of treating or preventingmyocardial dysfunction that occurs in dilated cardiomyopathy in asubject diagnosed with dilated cardiomyopathy, comprising administeringto the subject an effective amount of an inhibitor of CaMKII, wherebythe administration of the inhibitor treats or prevents dilatedcardiomyopathy or other structural heart disease in the subject.

[0016] The present invention provides a method of increasing myocardialcontractility in a subject diagnosed with dilated cardiomyopathy,comprising administering to the subject an effective amount of aninhibitor of CaMKII, whereby the administration of the inhibitorincreases myocardial contractility in the subject.

[0017] Further provided by the present invention is a method ofincreasing myocardial contractility in a subject diagnosed with cardiacdysfunction and/or decreased contractility following a myocardialinfarction, comprising administering to the subject an effective amountof an inhibitor of CaMKII, whereby the administration of the inhibitorincreases myocardial contractility in the subject.

[0018] Further provided by the present invention is a method ofincreasing myocardial contractility in a subject diagnosed withdecreased myocardial contractility following a myocardial infarction,comprising administering to the subject an effective amount of aninhibitor of CaMKII, whereby the administration of the inhibitorincreases myocardial contractility in the subject.

[0019] The present invention provides a method of identifying a compoundthat can treat structural heart disease, comprising: a) measuringcardiac contractility in an animal with structural heart disease; b)administering the compound to the animal of step (a); c) measuringcardiac contractility in the animal of step (b); and detecting anincrease in cardiac contractility in the animal of step (b) compared tocardiac contractility in the animal of step (a), whereby the detectionof an increase in cardiac contractility identifies a compound that cantreat structural heart disease.

[0020] The present invention provides a method of treating structuralheart disease in a subject, comprising administering to the subject aneffective amount of a compound identified by the method of the presentinvention.

[0021] The present invention provides a transgenic animal, whichexpresses a nucleic acid encoding an inhibitor of CaMKII.

[0022] The present invention also provides a transgenic animal whichexpresses a nucleic acid encoding a peptide comprising the peptide ofSEQ ID NO:8, which is referred to as AC3-C.

[0023] The present invention further provides a dual transgenic animal,which expresses a nucleic acid encoding an inhibitor of CaMKII and anucleic acid expressing calcineurin.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024]FIG. 1. A diagram of the domain structures of CaMK I, II, and IV,and the CaMKII inhibitory and control peptides expressed in the AC3-Iand AC3-C transgenic mice engineered for the present studies. CaMKII andIV both have a regulatory domain (black rectangle) that consists ofCaM-binding and autoinhibitory (AI) regions. The CaM binding region ofCaMKII (296-309, numbered according to CaMKII and marked by italics) isvery similar in CaMKIV (identical amino acids are underlined), andinhibitory peptides directed against the CaM-binding sequence in CaMKIIsimilarly inhibit activation of CaMKIV¹⁷. AC3-I is modeled on the AIregion of CaMKII that is centered around Thr 286 (marked with an arrow),and is dissimilar in CaMKII and CaMKIV. Neither the CaM-binding or AIregions of CaMKI are homologous to CaMKII or IV, but, in contrast to theAI region, inhibitory peptides directed against the CaM-binding regionof CaMKII or IV may nevertheless inhibit CaMKI because CaM-bindingdomains commonly share an a helical structure, but often lack primarysequence homology.¹³

[0025] FIGS. 2A-E. AC3-I mice have normal cardiac size and systolicfunction. A and B. Echocardiographic left ventricular dimensions are notsignificantly different between AC3-I mice and wild-type (WT) littermatecontrols in diastole (A) or systole (B). C and D. Interventricularseptal thickness is not different between AC3-I mice and WT littermatecontrols in diastole (C) or systole (D). E. Left ventricular fractionalshortening is not different between AC3-I and WT littermate control miceat baseline. Open bars are WT and black bars are AC3-I transgenic (TG)in all panels. The number of mice studied (n) in each line number(indicated in panel E) is the same in all panels.

[0026] FIGS. 3A-B. AC3-I mice have significantly reduced cardiac CaMKIIactivity (A) and significantly better left ventricular fractionalshortening after myocardial infarction surgery (B) than wild type (WT)littermate controls. CaMKII activity measurements are from whole hearthomogenates.

[0027]FIG. 4. Transgene expression in AC3-I and AC3-C mice by GFPWestern blots. Line numbers are indicated in parenthesis to indicateidentity in Western blots and corresponding quantitative phosphorimaging(data normalized to AC3-I line 5). The genetic identity of alltransgenic mice was confirmed by Southern analysis, but AC3-I line 3 didnot express the transgene.

[0028] FIGS. 5A-C. AC3-C transgenic mice (from line 1) develop dilatedcardiomyopathy. The left ventricular internal diameter (LVID) issignificantly greater in AC3-C (B) than AC3-I mice (B, **P<0.01, fromline 4) in diastole, indicating a dilated phenotype. The LVID shortenssignificantly less in AC3-C (B) than in AC3-I (A, ***P<0.001) miceduring systole, indicating reduced ventricular function. In contrast toLVID, the interventricular septum (IVS) and the left ventricularposterior wall (LVPW) are not significantly different between AC3-I andAC3-C mice in diastole or systole. C. Left ventricular fractionalshortening is significantly depressed in AC3-C compared to AC3-I mice(P<0.001). D. The heart rates are not different between AC3-I and AC3-Cmice.

[0029] FIGS. 6A-B. Total CaMKII activity is significantly reduced inventricular homogenates from AC3-I compared to AC3-C mice and wild-type(WT) littermates. A. Total CaMKII activity. B. Ca²⁺-independent fractionof CaMKII activity.

[0030] FIGS. 7A-D. CaMKII inhibition improves contractile function aftermyocardial infarction surgery. Echocardiographic measurements inunanesthetized mice 3 weeks after myocardial infarction surgery revealsignificantly preserved left ventricular function in mice with CaMKIIinhibition. A. Left ventricular (LV) fractional shortening issignificantly greater in mice treated daily with the CaMKII inhibitoryagent KN-93 (at 1 and 10 μmol/Kg body weight) and in AC3-I mice withtransgenically targeted CaMKII inhibition than in wild type littermatecontrols (WT) or in WT mice treated daily with the inactive KN-93congener KN-92 (30 μmol/Kg body weight). P<0.001 by ANOVA and asterisksindicate a significant difference compared to WT using a Bonferronicorrected t test. B. LV internal diameter (LVID) during diastole is amarker of LV chamber dilation. No significant differences in LVID duringdiastole were present between groups. C. LV posterior wall (LVPW) wallthickness in diastole is a measure of LV hypertrophy of thenon-infarcted wall. No significant differences in LVPW in diastole werepresent between groups. D. Heart rate was significantly slower in AC3-Imice with transgenically targeted CaMKII inhibition compared to allother groups.

[0031]FIG. 8. CaMKII inhibition reduces left ventricular dilation andimproves left ventricular contractile function in dilatedcardiomyopathy. Dual transgenic (AC3-I+/CAN+) have reduced leftventricular (LV) dilation and improved LV fractional shortening comparedto CAN+ transgenic mice.¹⁰ Echocardiographic measurements inunanesthetized calcineurin (CAN+) transgenic and interbred dualtransgenic AC3-I+/CAN+ mice show LV internal diameter (LVID) issignificantly reduced in diastole and the interventricular septum (IVS)and the LV posterior wall (LVPW) are increased in diastole inAC3-I+/CAN+ compared to CAN+ mice, indicating partial rescue of thedilated cardiomyopathy phenotype by transgenically targeted CaMKIIinhibition. Measurement parameters are the same for the top 4 panels:left ventricular internal diameter (LVID), interventricular septum(IVS), and left ventricular posterior wall (LVPW). Left ventricularsystolic function is significantly increased in the dual transgenicAC3-I+/CAN+ mice compared to the CAN+ mice, indicating improvedcontractile function by transgenically targeted cardiac CaMKIIinhibition. In contrast, there are no significant differences betweenheart rates in the AC3-I+/CAN+ and CAN+ transgenic animals. All micewere between 4-8 weeks of age.

[0032] FIGS. 9A-B. Strategy for interbreeding AC3-I or AC3-C andCalcineurin (CAN) transgenic mice. A. Boxes indicate breeding pairs. Theprimary comparison will be between CAN positive mice, but alternativecomparisons (that provide data shown in FIG. 8) are also shown. B. PCRresults reveal that interbreeding between AC3-I and CAN mice issuccessful and that dual transgenic mice can be identified with standardPCR methods.

DETAILED DESCRIPTION OF THE INVENTION

[0033] It must be noted that, as used in the specification and theappended claims, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “an inhibitor” includes multiple copies of theinhibitor and can also include more than one particular species ofinhibitor.

[0034] The present invention provides a method of treating or preventingmyocardial contractile dysfunction after a myocardial infarction in asubject diagnosed with myocardial contractile dysfunction after amyocardial infarction, comprising administering to the subject aneffective amount of an inhibitor of Calmodulin Kinase II (CaMKII),whereby the administration of the inhibitor treats or preventsmyocardial contractile dysfunction after myocardial infarction in thesubject In general, an “effective amount” of an inhibitor is that amountneeded to achieve the desired result or results. By “myocardialinfarction” is meant an ischemic injury to the heart in which part ofthe myocardium (heart muscle) has undergone necrosis or apoptosis, i.e.,programmed cell death. An “ischemic injury” means the damage orpotential damage to an organ or tissue that results from theinterruption of blood flow to the organ or tissue, i.e., an ischemicevent. As used throughout, by a “subject” is meant an individual. Thus,the “subject” can include domesticated animals, such as cats, dogs,etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.),laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.) andbirds. Preferably, the subject is a mammal such as a primate, and morepreferably, a human.

[0035] The subject can be a patient diagnosed as having a myocardialinfarction. The subject can be a patient diagnosed as havingpost-infarction cardiac dysfunction. The subject can be a patient whohas been diagnosed as having had a myocardial infarction who is, thus,at increased risk of developing post-infarction cardiac dysfunction.Further, the subject can be a patient diagnosed as having dilatedcardiomyopathy or symptoms of heart failure from any cause associatedwith a phenotype of cardiac chamber dilation and reduced myocardialcontractile function. The subject can be a patient diagnosed as havingreduced myocardial contractility. Methods of diagnosing myocardialinfarction, post-infarction cardiac dysfunction, reduced myocardialcontractility and dilated cardiomyopathy are well known in the art.Methods of distinguishing subjects having post-infarction contractiledysfunction or dilated cardiomyopathy from subjects having myocardialinfarction or having myocardial hypertrophy are well known in the art.It is recognized that a subject diagnosed with myocardial infarction, ora subject diagnosed with cardiac arrhythmia, or a subject diagnosed withmyocardial hypertrophy does not necessarily have dilated cardiomyopathyor reduced myocardial contractility.

[0036] An inhibitor of CaMKII can be any compound, composition or agentthat inhibits the activity or expression (e.g., the amount or thedisease-causing effect) of CaMKII. The compound can be a peptide ornon-peptide agent, including, for example, a nucleic acid that encodes apeptide inhibitor. Moreover, the agent can be an antisense nucleic acidthat inhibits expression of CaMKII in the heart (see GenBank accessionnumbers L13407 for isoform β3 and β2 from Hoch et al., Circ Res 1999 inFIG. 9 of this application)⁵ By “inhibit” is meant to restrict, holdback or reduce. Thus, an inhibitor is an agent that can, for example,reduce an activity of an enzyme or the amount of expression of anenzyme, or both. The inhibition can be reversible or irreversible.CaMKII activity in a subject or the amount of CaMKII in a subject can bereadily determined based on detection or measurement of a functionalresponse, for example as determined by echocardiography or by otherclinical parameters. Specific methods of measuring CaMKII activity in anon-human animal model are provided herein. Thus, it is routine toidentify compounds that inhibit CaMKII.

[0037] An example of an inhibitor of CaMKII is a peptide comprising thepeptide of SEQ ID NO:2, which is also referred to herein as AC3-I. Theinhibitor of the invention can consist of the peptide of SEQ ID NO:2.

[0038] Another example of an inhibitor of CaMKII is a peptide comprisingthe peptide of SEQ ID NO:4, which is CaM-KIIN. The inhibitor can be thefull-length CaM-KIIN (SEQ ID NO:4) and/or a fragment of the full-lengthpeptide; the fragment is called CaM-KIINtide (SEQ ID NO:6). CaMKIIN andCaM-KIINtide are described in Chang et al. PNAS (USA) 199895:10890-10895, which is herein incorporated by reference in itsentirety.

[0039] Because each of these is shown to inhibit CaMKII, it is expectedthat other peptides and polypeptides that contain it but includenon-essential amino acids will have similar activity. A non-essentialamino acid is an amino acid that will not affect the function of thepeptide or the way the peptide accomplishes that function (e.g., itssecondary structure or the ultimate result of the activity of thepeptide). Examples of non-essential amino acids in the present inventioninclude, but are not limited to, the amino acids comprising GFP, apeptide label that tags and identifies proteins or peptides forpurification

[0040] There are numerous other inhibitors of CaMKII that arecontemplated by the present invention, one of which is KN-93. KN-93, anon-peptide inhibitor of CaMKII, is described in WO 98/33491, which isherein incorporated by reference in its entirety for its teaching withregard to KN-93 and inhibitors of CaMKII.

[0041] The present invention further provides a method of treating orpreventing cardiac dysfunction following myocardial infarction in asubject, comprising administering to the subject an effective amount ofan inhibitor of CaMKII, whereby the administration of the inhibitortreats or prevents the cardiac dysfunction in the subject. By “cardiacdysfunction” is meant reduced contractile function of the blood pumpingchambers that results in the clinical condition of heart failure.Methods of diagnosing cardiac dysfunction following a myocardialinfarction in a subject are well known in the art.

[0042] In the method of treating or preventing cardiac dysfunctionfollowing a myocardial infarction in a subject, the inhibitor of CaMKIIcan be a peptide, for example, a peptide comprising the peptide of SEQID NO:2, or a peptide consisting of the peptide of SEQ ID NO:2. Theinhibitor of CaMKII can be a peptide comprising the peptide of SEQ IDNO:4 or a peptide consisting of the peptide of SEQ ID NO:4. Further, theinhibitor can be a peptide comprising the peptide SEQ ID NO:6 or apeptide consisting of the peptide of SEQ ID NO:6. The inhibitor can be anon-peptide inhibitor, for example, an inhibitor comprising the activeregion of KN-93 or it can be KN-93.

[0043] The present invention also provides a method of treating orpreventing dilated cardiomyopathy or any heart disease with thephenotypes of cardiac chamber dilation from any cause in a subject,comprising administering to the subject an effective amount of aninhibitor of CaMKII, whereby the administration of the inhibitor treatsdilated cardiomyopathy or reduces cardiac chamber dilation in thesubject. Methods of diagnosing dilated cardiomyopathy in a subject arewell known in the art.

[0044] In the method of treating or preventing dilated cardiomyopathy,the inhibitor of CaMKII can be a peptide, for example, a peptidecomprising the peptide of SEQ ID NO:2, or a peptide consisting of thepeptide of SEQ ID NO:2. The inhibitor of CaMKII can be a peptidecomprising the peptide of SEQ ID NO:4 or a peptide consisting of thepeptide of SEQ ID NO:4. Further, the inhibitor can be a peptidecomprising the peptide SEQ ID NO:6 or a peptide consisting of thepeptide of SEQ ID NO:6. The inhibitor can be a non-peptide inhibitor,for example, an inhibitor comprising the active region of KN-93 or itcan be KN-93.

[0045] The present invention provides a method of increasing myocardialcontractility in a subject diagnosed with dilated cardiomyopathy or anyheart disease with the phenotypes of cardiac chamber dilation or reducedcontractile function from any cause, comprising administering to thesubject an effective amount of an inhibitor of CaMKII, whereby theadministration of the inhibitor improves myocardial contractility in thesubject. Techniques for measuring cardiac contractility, for exampleechocardiography, radionucleotide angiography, and magnetic resonanceimaging are well known in the art. As used herein, “myocardialcontractility” is a measure of the contraction of the heart muscle, morespecifically, of the left ventricle. As shown in FIG. 7, AC3-I and KN-93increase myocardial contractility (a positive inotropic effect) withoutaffecting cardiac hypertrophy.

[0046] Further, the present invention provides a method of increasingmyocardial contractility in a subject diagnosed with myocardialinfarction, comprising administering to the subject an effective amountof an inhibitor of CaMKI, whereby the administration of the inhibitorimproves myocardial contractility in the subject.

[0047] The present invention also provides a method of increasingmyocardial contractility in a subject diagnosed with cardiac dysfunctionfollowing a myocardial infarction, comprising administering to thesubject an effective amount of an inhibitor of CaMKII, whereby theadministration of the inhibitor improves myocardial contractility in thesubject.

[0048] In the method of increasing myocardial contractility, theinhibitor of CaMKII can be a peptide, for example, a peptide comprisingthe peptide of SEQ ID NO:2, or a peptide consisting of the peptide ofSEQ ID NO:2. The inhibitor of CaMKII can be a peptide comprising thepeptide of SEQ ID NO:4 or a peptide consisting of the peptide of SEQ IDNO:4. Further, the inhibitor can be a peptide comprising the peptide SEQID NO:6 or a peptide consisting of the peptide of SEQ ID NO:6. Theinhibitor can be a non-peptide inhibitor, for example, an inhibitorcomprising the active region of KN-93 or it can be KN-93.

[0049] An inhibitor of CaMKII can be used to increase contractility ofthe myocardium without affecting myocardial hypertrophy, therebytreating a subject diagnosed with myocardial infarction, cardiacdysfunction following a myocardial infarction and/or dilatedcardiomyopathy.

[0050] The present invention provides a method of identifying a compoundthat can treat structural heart disease, comprising: a) measuringcardiac contractility in an animal with structural heart disease; b)administering the compound to the animal of step (a); c) measuringcardiac contractility in the animal of step (b); and d) detecting anincrease in cardiac contractility in the animal of step (b) compared tocardiac contractility in the animal of step (a), whereby the detectionof an increase in cardiac contractility identifies a compound that cantreat structural heart disease. In the method of this invention, theanimal with structural heart disease can be a transgenic animal thatexpresses AC3-C. Methods of measuring cardiac contractility are wellknown in the art and include, but are not limited to, echocardiography.Such methods include radionucleotide angiography, magnetic resonanceimaging, and left ventricular angiography.

[0051] The present invention provides a method of identifying a compoundthat can treat structural heart disease, comprising: a) measuring brainnatriuretic peptide in an animal with structural heart disease; b)administering the compound to the animal of step (a); c) measuring brainnatriuretic peptide in the animal of step (b); and d) detecting anincrease in brain natriuretic peptide in the animal of step (b) comparedto brain natriuretic peptide in the animal of step (a), whereby thedetection of an increase in brain natriuretic peptide identifies acompound that can treat structural heart disease. In the method of thisinvention, the animal with structural heart disease can be a transgenicanimal that expresses AC3-C.

[0052] Heart failure is a clinical syndrome that includes reducedexercise tolerance due to reduction in cardiac contraction and tissueoxygenation,7so exercise testing by treadmill or bicycle ergonometry,reduced tissue oxygen uptake, or increased plasma brain natriureticpeptide levels are all markers of heart failure severity. Valuesdenoting extreme and moderate impairment of myocardial contraction,exercise capacity, maximum oxygen consumption, and circulating brainnatriuretic peptide levels are well described and known to one skilledin the art of treating heart failure.⁷ Examples of structural heartdisease include, but are not limited to, myocardial infarction, cardiacdysfunction following myocardial infarction, reduced myocardialcontractility and dilated cardiomyopathy.

[0053] The present invention provides a method of treating myocardialinfarction, cardiac dysfunction following myocardial infarction and/ordilated cardiomyopathy in a subject, comprising administering to thesubject an effective amount of the compound identified by theabove-described method, whereby the administration of the compound tothe subject treats cardiac dysfunction following myocardial infarctionand/or dilated cardiomyopathy or patients with heart failure and dilatedcardiac chambers and reduced left ventricular contractility.

[0054] The present invention provides a transgenic animal that expressesa nucleic acid encoding an inhibitor of CaMKII. By “transgenic animal”is meant an animal in which all the cells of its body comprise anexogenous nucleic acid. In one example of a transgenic animal of theinvention, the transgene is specifically expressed in heart musclecells, driven by a heart cell specific promoter. This mouse wasengineered with a cardiac-specific α myosin heavy chain promoter(GenBank accession U71441). Methods of making transgenic animals arewell known in the art, and specifically exemplified herein.

[0055] In the transgenic animal of the present invention, the inhibitorof CaMKII that is expressed can be a peptide comprising the peptide ofSEQ ID NO:2. Moreover, the transgenic animal of the invention canexpress the peptide consisting of the peptide of SEQ ID NO:2. Recentreports have found at least 4 varieties of the CaMKII δ isoform inheart, 5 and AC3-I (SEQ ID NO:2) inhibits all CaMKII isoforms, due toconservation of the targeted regulatory domain.³ The AC3-I targeteddomain in CaMKII is dissimilar to analogous functional domains in otherCaMK types (i.e. CaMKI and CaMKIV, FIG. 1), and is virtually devoid ofactivity against protein kinases A and C.³

[0056] In the transgenic animal of the invention, the inhibitor ofCaMKII that is expressed can be a peptide comprising the peptide of SEQID NO:4. Moreover, the transgenic animal of the invention can expressthe peptide consisting of the peptide of SEQ ID NO:4.

[0057] The invention further provides a transgenic animal whichexpresses in heart muscle cells a nucleic acid encoding a peptidecomprising the peptide of SEQ ID NO:8, which is also referred to asAC3-C. As shown in FIGS. 4 and 5, AC3-I and AC3-C mice have similarexpression of transgene, but AC3-C mice exhibit cardiomyopathy, whereasAC3-I mice with CaMKII inhibition are normal. As shown in FIG. 6, theAC3-C mice have high total CaMKII activity as is seen in human patientswith cardiomyopathy. Thus, this transgenic animal can be used in thepresent method of identifying a compound that can treat structural heartdisease. Because AC3-C is believed to be inactive, these results may beexplained by an effect of the green fluorescent protein (GFP) moiety ofthe AC3-C-GFP transgene.⁶ This mouse is made following the basicprotocol for making the AC3-I transgenic mouse.

[0058] The present invention also provides a dual transgenic animal(AC3-I+/CAN+) which expresses in heart muscle cells a nucleic acidencoding AC3-I and a nucleic acid encoding calcineurin. The calcineurin(CAN+) transgenic mouse is a well accepted model of severe dilatedcardiomyopathy.¹⁰ CAN antagonists are known to ‘rescue’ thecardiomyopathy phenotype in a variety of mouse models,¹⁶ but CaMKIIinhibition has never been contemplated to improve cardiac function orstructure in these or any other models of cardiomyopathy. As exemplifiedin FIG. 8, the dual transgenic animal, which expresses an inhibitor ofCaMKII in addition to CAN, improves left ventricular function and showsreduced left ventricular dilation and hypertrophy when compared to CAN+animals. Thus, inhibition of CaMKII in this animal treats dilatedcardiomyopathy.

[0059] In the methods of the invention, an inhibitor of CaMKII can beadministered by known means. In a specific example, the peptideinhibitors are made cell membrane permeant. By cell “membrane permeant”is meant able to pass through the openings or interstices in amembrane¹⁶. This method uses a peptide sequence that is added to theinhibitory peptide, but myristoylation is another approach for making apeptide cell membrane permeant.

[0060] The compositions of the present invention can also beadministered in vivo in a pharmaceutically acceptable carrier. By“pharmaceutically acceptable” is meant a material that is notbiologically or otherwise undesirable, i.e., the material may beadministered to a subject, along with the composition, without causingany undesirable biological effects or interacting in a deleteriousmanner with any of the other components of the pharmaceuticalcomposition in which it is contained. The carrier would naturally beselected to minimize any degradation of the active ingredient and tominimize any adverse side effects in the subject, as would be well knownto one of skill in the art.

[0061] The compositions may be administered orally, parenterally (e.g.,intravenously, intramuscularly, intrathecally, intraarterially and byintraperitoneal injection), transdermally, extracorporeally, topicallyor the like, although topical intranasal administration oradministration by inhalant is typically preferred. As used herein,“topical intranasal administration” means delivery of the compositionsinto the nose and nasal passages through one or both of the nares andcan comprise delivery by a spraying mechanism or droplet mechanism, orthrough aerosolization of the therapeutic agent. Delivery can also bedirectly to any part of the lower respiratory tract (e.g., trachea,bronchi and lungs) via intubation. The exact amount of the compositionsrequired will vary from subject to subject, depending on the species,age, weight and general condition of the subject, the severity of thecondition being treated, the particular composition used, its mode ofadministration and the like. Thus, it is not possible to specify anexact amount for every composition. However, an appropriate amount canbe determined by one of ordinary skill in the art using only routineexperimentation given the teachings herein.

[0062] Parenteral administration of the composition, if used, isgenerally characterized by injection. Injectables can be prepared inconventional forms, either as liquid solutions or suspensions, solidforms suitable for solution of suspension in liquid prior to injection,or as emulsions. A more recently revised approach for parenteraladministration involves use of a slow release or sustained releasesystem such that a constant dosage is maintained. See, e.g., U.S. Pat.No. 3,610,795, which is incorporated by reference herein.

[0063] The materials may be in solution, suspension (for example,incorporated into microparticles, liposomes, or cells). These may betargeted to a particular cell type via antibodies, receptors, orreceptor ligands. The following references are examples of the use ofthis technology to target specific proteins to tumor tissue (Senter, etal., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K. D., Br. J.Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703,(1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, etal., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz andMcKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al.,Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as “stealth”and other antibody conjugated liposomes (including lipid mediated drugtargeting to colonic carcinoma), receptor mediated targeting of DNAthrough cell specific ligands, lymphocyte directed tumor targeting, andhighly specific therapeutic retroviral targeting of murine glioma cellsin vivo. The following references are examples of the use of thistechnology to target specific proteins to tumor tissue (Hughes et al.,Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang,Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general,receptors are involved in pathways of endocytosis, either constitutiveor ligand induced. These receptors cluster in clathrin-coated pits,enter the cell via clathrin-coated vesicles, pass through an acidifiedendosome in which the receptors are sorted, and then either recycle tothe cell surface, become stored intracellularly, or are degraded inlysosomes. The internalization pathways serve a variety of functions,such as nutrient uptake, removal of activated proteins, clearance ofmacromolecules, opportunistic entry of viruses and toxins, dissociationand degradation of ligand, and receptor-level regulation. Many receptorsfollow more than one intracellular pathway, depending on the cell type,receptor concentration, type of ligand, ligand valency, and ligandconcentration. The molecular and cellular mechanisms ofreceptor-mediated endocytosis have been reviewed (Brown and Greene, DNAand Cell Biology 10:6, 399-409 (1991)).

[0064] The compositions of the present invention can include a nucleicacid encoding an inhibitor or can include a CaMKII antisense nucleicacid. The disclosed compositions can be delivered to the target cells ina variety of ways. For example, the compositions can be deliveredthrough electroporation, or through lipofection, or through calciumphosphate precipitation. The delivery mechanism chosen will depend inpart on the type of cell targeted and whether the delivery is occurringfor example in vivo or in vitro.

[0065] Thus, the compositions can comprise, in addition to the disclosedcompositions or vectors for example, lipids such as liposomes, such ascationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionicliposomes. Liposomes can further comprise proteins to facilitatetargeting a particular cell, if desired. Administration of a compositioncomprising a compound and a cationic liposome can be administered to theblood afferent to a target organ or inhaled into the respiratory tractto target cells of the respiratory tract. Regarding liposomes, see,e.g., Brigham et al. Am. J. Resp. Cell. Mol. Biol. 1:95-100 (1989);Felgner et al. Proc. Natl. Acad. Sci USA 84:7413-7417 (1987); U.S. Pat.No. 4,897,355. Furthermore, the compound can be administered as acomponent of a microcapsule that can be targeted to specific cell types,such as macrophages, or where the diffusion of the compound or deliveryof the compound from the microcapsule is designed for a specific rate ordosage.

[0066] In the methods described above which include the administrationand uptake of exogenous DNA into the cells of a subject (i.e., genetransduction or transfection), delivery of the compositions to cells canbe via a variety of mechanisms. As one example, delivery can be via aliposome, using commercially available liposome preparations such asLIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.),SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (PromegaBiotec, Inc., Madison, Wis.), as well as other liposomes developedaccording to procedures standard in the art. In addition, the nucleicacid or vector of this invention can be delivered in vivo byelectroporation, the technology for which is available from Genetronics,Inc. (San Diego, Calif.) as well as by means of a SONOPORATION machine(ImaRx Pharmaceutical Corp., Tucson, Ariz.).

[0067] Nucleic acids that are delivered to cells which are to beintegrated into the host cell genome, typically contain integrationsequences. These sequences are often viral related sequences,particularly when viral based systems are used. These viral integrationsystems can also be incorporated into nucleic acids which are to bedelivered using a non-nucleic acid based system of delivery, such as aliposome, so that the nucleic acid contained in the delivery system canbe come integrated into the host genome. As used herein, “nucleic acid”includes single- or double-stranded molecules which may be DNA,comprised of the nucleotide bases A, T, C, G or RNA, comprised of thebases A, U (substitutes for T), C and G. The nucleic acid may representa coding strand or its complement. Nucleic acids may be identical insequence to the portion of the sequence which is naturally occurring ormay include alternative codons which encode the same amino acid as thatwhich is found in the naturally occurring sequence. Furthermore, nucleicacids can include codons which represent conservative substitutions ofamino acids as are well known in the art.

[0068] Other general techniques for integration into the host genomeinclude, for example, systems designed to promote homologousrecombination with the host genome. These systems typically rely onsequence flanking the nucleic acid to be expressed that has enoughhomology with a target sequence within the host cell genome thatrecombination between the vector nucleic acid and the target nucleicacid takes place, causing the delivered nucleic acid to be integratedinto the host genome. Those of skill in the art know these systems andthe methods necessary to promote homologous recombination.

[0069] As described above, the compositions can be administered in apharmaceutically acceptable carrier and can be delivered to the cells ofthe subject in vivo and/or ex vivo by a variety of mechanisms well knownin the art (e.g., uptake of naked DNA, liposome fusion, intramuscularinjection of DNA via a gene gun, endocytosis and the like). If ex vivomethods are employed, cells or tissues can be removed and maintainedoutside the body according to standard protocols well known in the art.The compositions can be introduced into the cells via any gene transfermechanism, such as, for example, calcium phosphate mediated genedelivery, electroporation, microinjection or proteoliposomes. Thetransduced cells can then be infused (e.g., in a pharmaceuticallyacceptable carrier) or homotopically transplanted back into the subjectper standard methods for the cell or tissue type. Standard methods areknown for transplantation or infusion of various cells into a subject.

[0070] The inhibitor can be administered in any dose that is effectiveto inhibit CaMKII activity or amount. As noted above, detection of areduction in CaMKII activity or amount is well within the skill of thepractitioner. More specifically, the inhibitor can be administered in adose of from about 0.05 mg to about 5.0 mg per kilogram of body weight.The inhibitor can, alternatively, be administered in a dose of fromabout 0.3 mg to about 3.0 mg per kilogram of body weight.

[0071] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow the compositions and/or methods claimed herein are made andevaluated, and are intended to be purely exemplary of the invention andare not intended to limit the scope of what the inventors regard astheir invention. Efforts have been made to ensure accuracy with respectto numbers (e.g., amounts, temperature, etc.), but some errors anddeviations should be accounted for. The present invention is moreparticularly described in the following examples which are intended asillustrative only since numerous modifications and variations thereinwill be apparent to those skilled in the art.

EXAMPLES

[0072] AC3-I transgenic mice: The AC3-I mice were generated by synthesisof a minigene based on the peptide sequence for AC3-I (FIG. 1). A‘minigene’ encoding AC3-I (KKALHRQEAVDCL), ² a CaMKII inhibitory peptidewas constructed with these complementary oligonucleotides:GATCAAAAAAGCCCTTCACCGCCAGGAGGCAGTTGAC (SEQ ID NO:1)TGCCTTGCTTTTTTCGGGAAGTGGCGGTCCTCCGTCA ACTGACGGAACGCTAG,

[0073] and a minigene was similarly constructed for a related, inactivecontrol peptide, AC3-C (KKALHAQERVDCL) using the following complementaryoligonucleotides: GATCAAAAAAGCCCTTCACGCACAGGAGCGCGTTGAC (SEQ ID NO:7)TGCCTTGCTTTTTTCGGGAAGTGCGTGTCCTCGCGCA ACTGACGGAACGCTAG

[0074] The minigene was inserted in frame with the EGFP into the BspEIsite of pEGFP-Cl (Clontech), which places the EGFP at the N-terminus ofthe peptide. The AC3-I minigene includes a Kozak consensus translationalstart site. It was then sequenced and expressed in HEK293 cells to showgreen fluorescence. Previous studies indicated that an AC3-I-GST-MTSfusion peptide retained full CaMKII inhibitory potency against asynthetic CaMKII substrate (AC3-I-GST-MTS IC₅₀=0.4 μM; AC3-I IC₅₀ 0.5μM), suggesting that the AC3-I-GFP protein would also retain CaMKIIinhibitory activity. The 800 bp AC3-I-GFP sequence was then amplifiedusing PCR, purified and subcloned into the SalI site of a pBluescriptvector containing the α-MHC promoter vector (GenBank accession U71441)and the human growth hormone (HGH) poly A tail (developed by Dr. J.Robbins). The construct was verified by sequencing and expressed in amurine atrial tumor cell line (HL1), which also showed greenfluorescence. Murine embryonic stem cells were injected with thelinearized DNA (2 ng/ml) in the Vanderbilt Transgenic Mouse corefacility and implanted in B6D2 pseudo-pregnant females.

[0075] The AC3-I is linked to green fluorescent protein (GFP) to revealhomogenous expression throughout the heart when histologic sections areexamined under a microscope. These AC3-I mice are viable and have normalbasal cardiac size and function (FIG. 2). However, hearts from thesemice have significantly reduced total cardiac CaMKII activity (FIG.3(A)), and these mice have significantly less impairment of cardiacfunction after experimental myocardial infarction compared to wild typelittermate controls (FIG. 3(B)). These findings indicate that CaMKIIactivity is a novel signal for cardiac dysfunction after myocardialinfarction and are the basis for our claim to treat patients withmyocardial infarction by a method of CaMKII inhibition.

[0076] AC3-I+/CAN+ transgeneic mice: Dual transgenic mice were interbredusing the strategy schematized in FIG. 9. For genotyping of the firstgeneration, tail biopsies were taken at 3 weeks of age and wereincubated overnight at 55° C. in 0.5 Mg/ml protease K, 50 mM Tris (pH8.0), 100 mM EDTA, and 0.5% SDS. Genomic DNA was precipitated with anequal volume of isopropanol after performing phenol/chloroform/isoamylalcohol (25:24:1) extraction twice. DNA was dissolved overnight in 50 μlof {fraction (1/10)} TE (pH 8.0) with 2.5 μg RNAase A. 15 μg of genomicDNA was completely digested with EcoRI and digested DNA was separated ona 0.8% agarose gel in 1×TAE. After electrophoresis, the gel wasincubated in 0.25N HCL for 30 min, neutralized in 0.5M NaOH-1.5M NaClfor 15 min twice and equilibrated in 0.5M Tris-1.5M NaCl for 15 mintwice. The gel was blotted overnight on MSI nylon transfer membrane(Micron separations Inc. Westborought, Mass.) and the filter wasUV-crosslinked. GFP-AC3I or GFP-AC3C DNA fragments were labeled with ³²Pby random oligonucleotide priming (Stratagene, La Jolla, Calif.) as aprobe. Hybridization was carried out overnight at 42° C. in the presenceof 50% Formamide, 5×SSPE, 5× Denhardt's solution, 0.1% SDS, and 1001g/ml denatured, fragmented salmon sperm DNA. The filter was washed in0.5×SSC-0.1% SDS for 30 min at 65° C., followed by a wash in0.1×SSC-0.1% SDS for 20 min twice at 65° C. The filter was exposed toKodak autoradiography film and developed afterwards.

[0077] Routine screening of transgenic mice, after the first generation,was done by PCR (see FIG. 9). Two primers served to amplify a 442-bpregion of the human growth hormone (hGH) gene at 3′ end. The sequencesof the primers are 5′-Hgh (5′-(SEQ ID NO:9) GTCTATTCGGGAACCAAGCT-3′) and3′-Hgh (5′-(SEQ ID NO:10) ACAGGCATCTACTGAGTGGACC-3′). 100 ng purifiedgenomic DNA was mixed with 200 pM of each primer and amplified accordingto the following protocol: 95° C. for 5 min, followed by 30 cyclesconsisting of 95° C. for 45 seconds, 50° C. for 45 seconds, 72° C. for 1min and a final 7 min elongation at 72° C. All samples were run on a1.5% agarose gel in the presence of 0.5 μg/ml ethidium bromide andstained DNA bands were visualized under UV light. New primer sets weredeveloped for the interbred dual transgenic mice (FIG. 9) and theseallow differentiation of dual and single transgenic animals.

[0078] Echocardiography: Echocardiography is performed using a HewlettPackard Sonos 5500 (fully dedicated to murine studies) and a speciallydeveloped 12 MHz probe. Cardiac dimensions are obtained from 2-D-guidedM mode images and are read off line by blinded, independent readersusing short axis and a parasternal long-axis views with the leading edgemethod. Animals can be lightly sedated with pentobarbital (15 mg/Kg,i.p.); however, measurements without anesthesia, where the mice undergoa brief ‘training’ period to acclimate to the procedure, can be made.Approximately 90% of mice can be trained to undergo echocardiographicstudies without the cardiodepressant effects of anesthesia. Measurementsare averaged over 3 consecutive beats from the LV posterior wall (LVPW),the interventricular septum (IVS), and the LV internal diameter (LVID).Fractional shortening (FS) is used to estimate systolic function and iscomputed according to the formulaFS=(LVIDdiastole-LVIDsystole)/LVIDdiastole×100.

[0079] Myocardial infarction surgery: Surgery is routinely performed atthe Vanderbilt mouse physiology core laboratory and is essentiallyidentical to other published reports ¹⁸ ⁸.¹⁴ In brief, mice areanesthetized (pentobarbital 33 μg/g and ketamine 33 μg/g, i.p.) andplaced on a rodent respirator (tidal vol. 0.5 ml, rate 120 breaths/min),the chest opened with a left parasternal thoracotomy, and the region ofthe mid-left anterior descending artery ligated with 8-0 suture. Thechest is closed (running 5-0 suture), and the animal weaned from therespirator. Wild-type mice typically develop heart failure, as assessedby increased lung wet:dry weights. Sham operations omit the ligationstep. Approximately 75% of animals survive surgery with a successfullyinfarcted anterior wall.

[0080] CaMKII activity assays: CaMKII activity was assayed in AC3-I miceand littermate controls from whole heart (ventricular) homogenate withsyntide 2-a synthetic substrate with ˜50 fold selectivity for CaMKIIover CaMKIV,⁹ using our previously published methods.¹

[0081] Dilated cardiomyopathy: Compelling data are presented onprevention of dilated cardiomyopathy by CaMKII inhibition. A controltransgenic mouse that expresses GFP linked to AC3-C (an inactivecongener of AC3-I) was made. This mouse develops rather severe dilatedcardiomyopathy that is absent in the AC3-I-GFP mouse, and has very highlevels of CaMKII activity (FIG. 5).

[0082] Throughout this application, various publications are referenced.The disclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

[0083] It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

REFERENCES

[0084] 1. Anderson M., Braun A., Wu Y., Lu T., Schulman H., Sung R.KN-93, an inhibitor of multifunctional Ca++/calmodulin-dependent proteinkinase, decreases early afterdepolarizations in rabbit heart. J PharmExp Ther 1998;287:996-1006.

[0085] 2. Braun A., Schulman H. A non-selective cation current activatedvia the multifunctional Ca(2+)-calmodulin-dependent protein kinase inhuman epithelial cells. J Physiol 1995;488:37-55.

[0086] 3. Braun A., Schulman H. The multifunctionalcalcium/calmodulin-dependent protein kinase: from form to function. AnnRev Physiol 1995;57:417-445.

[0087] 4. Gottlieb S., McCarter R., Vogel R. Effect of beta-blockade onmortality among high-risk and low-risk patients after myocardialinfarction. [see comments]. New England Journal of Medicine1998;339:489-497.

[0088] 5. Hoch B., Meyer R., Hetzer R., Krause E., Karczewski P.Identification and expression of delta-isoforms of the multifunctionalCa2+/calmodulin-dependent protein kinase in failing and nonfailing humanmyocardium. Circulation Research 1999;84:713-721.

[0089] 6. Huang W., Aramburu J., Douglas P., Izumo S. Transgenicexpression of green fluorescence protein can cause dilatedcardiomyopathy. Nat Med 2000;6:482-483.

[0090] 7. Hunt S., Baker D., Chin M., Cinquegrani M., Feldman A.,Francis G., Ganiats T., Goldstein S., Gregoratos G., Jessup M., NobleR., Paclcer M., Silver M., Stevenson L., Gibbons R., Antman E., AlpertJ., Faxon D., Fuster V., Jacobs A., Hiratzka L., Russell R., Smith S.,Jr., American College of Cardiology/American Heart Association. ACC/AHAguidelines for the evaluation and management of chronic heart failure inthe adult: executive summary. A report of the American College ofCardiology/American Heart Association Task Force on Practice Guidelines(Committee to revise the 1995 Guidelines for the Evaluation andManagement of Heart Failure). Journal of the American College ofCardiology 2001;38:2101-2113.

[0091] 8. Kinugawa S., Tsutsui H., Hayashidani S., Ide T., Suematsu N.,Satoh S., Utsumi H., Takeshita A. Treatment with dimethylthioureaprevents left ventricular remodeling and failure after experimentalmyocardial infarction in mice: role of oxidative stress. CirculationResearch 2000;87:392-398.

[0092] 9. Miyano O., Kameshita I., Fujisawa H. Purification andcharacterization of a brain-specific multifunctionalcalmodulin-dependent protein kinase from rat cerebellum. J Biol Chem1992;267:1198-1203.

[0093] 10. Molkentin J., Lu J., Antos C., Markham B., Richardson J.,Robbins J., Grant S., Olson E. A calcineurin-dependent transcriptionalpathway for cardiac hypertrophy. Cell 1998;93:215-228.

[0094] 11. Pfeffer J., Fischer T., Pfeffer M. Angiotensin-convertingenzyme inhibition and ventricular remodeling after myocardialinfarction. Ann Rev Physiol 1995;57:805-826.

[0095] 12. Pitt B., Zannad F., Remme W., Cody R., Castaigne A., PerezA., Palensky J., Wittes J. The effect of spironolactone on morbidity andmortality in patients with severe heart failure. Randomized AldactoneEvaluation Study Investigators. New England Journal of Medicine1999;341:709-717.

[0096] 13. Rhoads A., Friedberg F. Sequence motifs for calmodulinrecognition. FASEB 1997;11:331-340.

[0097] 14. Sam F., Sawyer D., Chang D., Eberli F., Ngoy S., Jain M.,Amin J., Apstein C., Colucci W. Progressive left ventricular remodelingand apoptosis late after myocardial infarction in mouse heart. AmericanJournal of Physiology—Heart & Circulatory Physiology 2000;279:H422-H428.

[0098] 15. Spencer F., Meyer T., Goldberg R., Yarzebski J., Hatton M.,Lessard D., Gore J. Twenty year trends (1975-1995) in the incidence,in-hospital and long-term death rates associated with heart failurecomplicating acute myocardial infarction: a community-wide perspective.J Am Coll Cardiol 1999;34:1378-1387.

[0099] 16. Sussman M., Lim H., Gude N., Taigen T., Olson E., Robbins J.,Colbert, M C, Gualberto A., Wieczorek D., Molkentin J. Prevention ofcardiac hypertrophy in mice by calcineurin inhibition [see comments].Science 1998;281:1690-1693.

[0100] 17. Tokumitsu H., Brickey D., Glod J., Hidaka H., Silcela J.,Soderling T. Activation mechanisms for Ca2+/calmodulin-dependent proteinkinase IV. Identification of a brain CaM-kinase IV kinase. J Biol Chem1994;269:28640-28647.

[0101] 18. Trueblood N., Xie Z., Communal C., Sam F., Ngoy S., Liaw L.,Jenkins A., Wang J., Sawyer D., Bing O., Apstein C., Colucci W., SinghK. Exaggerated left ventricular dilation and reduced collagen depositionafter myocardial infarction in mice lacking osteopontin. CirculationResearch 2001;88:1080-1087.

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[0104] 21. Wu Y. Calmodulin kinase II and arrhythmias in a mouse modelof cardiac hypertrophy. Circ 2002;106:1288-1293.

1 10 1 90 DNA Artificial Sequence Description of Artificial Sequence;Note = Synthetic Construct 1 gatcaaaaaa gcccttcacc gccaggaggc agttgactgccttgcttttt tcgggaagtg 60 gcggtcctcc gtcaactgac ggaacgctag 90 2 13 PRTArtificial Sequence Description of Artificial Sequence; Note = SyntheticConstruct 2 Lys Lys Ala Leu His Arg Gln Glu Ala Val Asp Cys Leu 1 5 10 3255 DNA Artificial Sequence Description of Artificial Sequence; Note =Synthetic Construct 3 cggctcccct gctgagctag ggccggtccg gcagtcagcctctgcccgtg ccccgccgca 60 gtccctagcc cgcccggtgc ccgccgcctg caggacaccaactccttctt cgctggcaac 120 caggccaagc ggccccccaa gctgggccag atcggccgagccaagagagt ggtgatcgag 180 gatgaccgga tagacgacgt gctgaagggg atgggggagaagcctccgtc cggagtgtag 240 acgcgccggc tctgg 255 4 79 PRT ArtificialSequence Description of Artificial Sequence; Note = Synthetic Construct4 Met Ser Glu Ile Leu Pro Tyr Gly Glu Asp Lys Met Gly Arg Phe Gly 1 5 1015 Ala Asp Pro Glu Gly Ser Asp Leu Ser Phe Ser Cys Arg Leu Gln Asp 20 2530 Thr Asn Ser Phe Phe Ala Gly Asn Gln Ala Lys Arg Pro Pro Lys Leu 35 4045 Gly Gln Ile Gly Arg Ala Lys Arg Val Val Ile Glu Asp Asp Arg Ile 50 5560 Asp Asp Val Leu Lys Gly Met Gly Glu Lys Pro Pro Ser Gly Val 65 70 755 129 DNA Artificial Sequence Description of Artificial Sequence; Note =Synthetic Construct 5 aagcggcccc ccaagctggg ccagatcggc cgagccaagagagtggtgat cgaggatgac 60 cggatagacg acgtgctgaa ggggatgggg gagaagcctccgtccggagt gtagacgcgc 120 cggctctgg 129 6 27 PRT Artificial SequenceDescription of Artificial Sequence; Note = Synthetic Construct 6 Lys ArgPro Pro Lys Leu Gly Gln Ile Gly Arg Ala Lys Arg Val Val 1 5 10 15 IleGlu Asp Asp Arg Ile Asp Asp Val Leu Lys 20 25 7 90 DNA ArtificialSequence Description of Artificial Sequence; Note = Synthetic Construct7 gatcaaaaaa gcccttcacg cacaggagcg cgttgactgc cttgcttttt tcgggaagtg 60cgtgtcctcg cgcaactgac ggaacgctag 90 8 13 PRT Artificial SequenceDescription of Artificial Sequence; Note = Synthetic Construct 8 Lys LysAla Leu His Ala Gln Glu Arg Val Asp Cys Leu 1 5 10 9 20 DNA ArtificialSequence Description of Artificial Sequence; Note = Synthetic Construct9 gtctattcgg gaaccaagct 20 10 22 DNA Artificial Sequence Description ofArtificial Sequence; Note = Synthetic Construct 10 acaggcatct actgagtggacc 22

What is claimed is:
 1. A method of treating or preventing myocardialcontractile dysfunction after myocardial infarction in a subject,comprising administering to the subject an effective amount of aninhibitor of Calmodulin Kinase II (CaMKII), whereby the administrationof the inhibitor treats or prevents post-myocardial infarctionmyocardial contractile dysfunction in the subject.
 2. The method ofclaim 1, wherein the inhibitor of CaMKII is a peptide comprising thepeptide of SEQ ID NO:2.
 3. The method of claim 2, wherein the inhibitoris the peptide of SEQ ID NO:
 2. 4. The method of claim 1, wherein theinhibitor of CaMKII is a peptide comprising the peptide of SEQ ID NO:4.5. The method of claim 4, wherein the inhibitor is the peptide of SEQ IDNO:4.
 6. The method of claim 1, wherein the inhibitor of CaMKII is apeptide comprising the peptide of SEQ ID NO:6.
 7. The method of claim 6,wherein the inhibitor is the peptide of SEQ ID NO:6.
 8. The method ofclaim 1, wherein the inhibitor is KN-93.
 9. The method of claim 1,wherein the inhibitor is administered in a dose of from about 0.05 mg toabout 5.0 mg per kilogram of body weight.
 10. The method of claim 1,wherein the inhibitor is administered in a dose of from about 0.3 mg toabout 3.0 mg per kilogram of body weight.
 11. A method of treating orpreventing dilated cardiomyopathy in a subject diagnosed with dilatedcardiomyopathy, comprising administering to the subject an effectiveamount of an inhibitor of Calmodulin Kinase II (CaMKII), whereby theadministration of the inhibitor treats or prevents dilatedcardiomyopathy in the subject.
 12. The method of claim 11, wherein theinhibitor of CaMKII is a peptide comprising the peptide of SEQ ID NO:2.13. The method of claim 12, wherein the inhibitor is the peptide of SEQID NO:
 2. 14. The method of claim 11, wherein the inhibitor of CaMKII isa peptide comprising the peptide of SEQ ID NO:4.
 15. The method of claim14, wherein the inhibitor is the peptide of SEQ ID NO:4.
 16. The methodof claim 11, wherein the inhibitor of CaMKII is a peptide comprising thepeptide of SEQ ID NO:6.
 17. The method of claim 16, wherein theinhibitor is the peptide of SEQ ID NO:6.
 18. The method of claim 11,wherein the inhibitor is KN-93.
 19. The method of claim 11, wherein theinhibitor is administered in a dose of from about 0.05 mg to about 5.0mg per kilogram of body weight.
 20. The method of claim 11, wherein theinhibitor is administered in a dose of from about 0.3 mg to about 3.0 mgper kilogram of body weight.
 21. A method of increasing myocardialcontractility in a subject diagnosed with dilated cardiomyopathy,comprising administering to the subject an effective amount of aninhibitor of CaMKII, whereby the administration of the inhibitorincreases myocardial contractility in the subject.
 22. The method ofclaim 21, wherein the inhibitor of CaMKII is a peptide comprising thepeptide of SEQ ID NO:2.
 23. The method of claim 22, wherein theinhibitor is the peptide of SEQ ID NO:
 2. 24. The method of claim 21,wherein the inhibitor of CaMKII is a peptide comprising the peptide ofSEQ ID NO:4.
 25. The method of claim 24, wherein the inhibitor is thepeptide of SEQ ID NO:4.
 26. The method of claim 21, wherein theinhibitor of CaMKII is a peptide comprising the peptide of SEQ ID NO:6.27. The method of claim 26, wherein the inhibitor is the peptide of SEQID NO:6.
 28. The method of claim 21, wherein the inhibitor is KN-93. 29.The method of claim 21, wherein the inhibitor is administered in a doseof from about 0.05 mg to about 5.0 mg per kilogram of body weight. 30.The method of claim 21, wherein the inhibitor is administered in a doseof from about 0.3 mg to about 3.0 mg per kilogram of body weight.
 31. Amethod of increasing myocardial contractility in a subject diagnosedwith decreased myocardial contractility, comprising administering to thesubject an effective amount of an inhibitor of CaMKII, whereby theadministration of the inhibitor increases myocardial contractility inthe subject.
 32. The method of claim 31, wherein the inhibitor of CaMKIIis a peptide comprising the peptide of SEQ ID NO:2.
 33. The method ofclaim 32, wherein the inhibitor is the peptide of SEQ ID NO:
 2. 34. Themethod of claim 31, wherein the inhibitor of CaMKII is a peptidecomprising the peptide of SEQ ID NO:4.
 35. The method of claim 34,wherein the inhibitor is the peptide of SEQ ID NO:4.
 36. The method ofclaim 31, wherein the inhibitor of CaMKII is a peptide comprising thepeptide of SEQ ID NO:6.
 37. The method of claim 36, wherein theinhibitor is the peptide of SEQ ID NO:6.
 38. The method of claim 31,wherein the inhibitor is KN-93.
 39. The method of claim 31, wherein theinhibitor is administered in a dose of from about 0.05 mg to about 5.0mg per kilogram of body weight.
 40. The method of claim 31, wherein theinhibitor is administered in a dose of from about 0.3 mg to about 3.0 mgper kilogram of body weight.
 41. A method of increasing myocardialcontractility in a subject diagnosed with cardiac structural dysfunctionfollowing a myocardial infarction, comprising administering to thesubject an effective amount of an inhibitor of CaMKII, whereby theadministration of the inhibitor increases myocardial contractility inthe subject.
 42. The method of claim 41, wherein the inhibitor of CaMKIIis a peptide comprising the peptide of SEQ ID NO:2.
 43. The method ofclaim 42, wherein the inhibitor is the peptide of SEQ ID NO:2.
 44. Themethod of claim 41, wherein the inhibitor of CaMKII is a peptidecomprising the peptide of SEQ ID NO:4.
 45. The method of claim 44,wherein the inhibitor is the peptide of SEQ ID NO:4.
 46. The method ofclaim 41, wherein the inhibitor of CaMKII is a peptide comprising thepeptide of SEQ ID NO:6.
 47. The method of claim 46, wherein theinhibitor is the peptide of SEQ ID NO:6.
 48. The method of claim 41,wherein the inhibitor is KN-93.
 49. The method of claim 41, wherein theinhibitor is administered in a dose of from about 0.05 mg to about 5.0mg per kilogram of body weight.
 50. The method of claim 41, wherein theinhibitor is administered in a dose of from about 0.3 mg to about 3.0 mgper kilogram of body weight.
 51. A transgenic animal which expresses anucleic acid encoding an inhibitor of CaMKII.
 52. The transgenic animalof claim 51, wherein the inhibitor of CaMKII is a peptide comprising thepeptide of SEQ ID NO:2.
 53. The transgenic animal of claim 52, whereinthe inhibitor is the peptide of SEQ ID NO:2.
 54. The transgenic animalof claim 51, wherein the inhibitor of CaMKII is a peptide comprising thepeptide of SEQ ID NO:4.
 55. The transgenic animal of claim 54, whereinthe inhibitor is the peptide of SEQ ID NO:4.
 56. The transgenic animalof claim 51, wherein the inhibitor of CaMKII is a peptide comprising thepeptide of SEQ D NO:6.
 57. The transgenic animal of claim 56, whereinthe inhibitor is the peptide of SEQ ID NO:6.
 58. A dual transgenicanimal which expresses a nucleic acid encoding an inhibitor of CaMKIIand a nucleic acid encoding calcineurin.
 59. The dual transgenic animalof claim 58, wherein the inhibitor of CaMKII is a peptide comprising thepeptide of SEQ ID NO:2.
 60. The dual transgenic animal of claim 59,wherein the inhibitor is the peptide of SEQ ID NO:2.
 61. The dualtransgenic animal of claim 58, wherein the inhibitor of CaMKII is apeptide comprising the peptide of SEQ ID NO:4.
 62. The dual transgenicanimal of claim 61, wherein the inhibitor is the peptide of SEQ ID NO:4.63. The dual transgenic animal of claim 58, wherein the inhibitor ofCaMKII is a peptide comprising the peptide of SEQ ID NO:6.
 64. The dualtransgenic animal of claim 63, wherein the inhibitor is the peptide ofSEQ ID NO:6.
 65. A transgenic animal which expresses a nucleic acidencoding a peptide comprising the peptide of SEQ ID NO:8.
 66. Thetransgenic animal of claim 65, wherein the transgenic animal expresses anucleic acid encoding the peptide of SEQ ID NO:8.
 67. A method ofidentifying a compound that can treat structural heart disease,comprising: a) measuring cardiac contractility in an animal of claim 51;b) administering the compound to the animal of claim 51; c) measuringcardiac contractility in the animal of step (b); and d) detecting anincrease in cardiac contractility in the animal of step (b) compared tocardiac contractility in the animal of step (a), whereby the detectionof an increase in cardiac contractility identifies a compound that cantreat structural heart disease.
 68. The method of claim 67, wherein thestructural heart disease can be at least one of dilated cardiomyopathyor myocardial contractile dysfunction.
 69. A method of identifying acompound that can treat structural heart disease, comprising: a)measuring cardiac contractility in an animal of claim 58; b)administering the compound to the animal of claim 58; c) measuringcardiac contractility in the animal of step (b); and d) detecting anincrease in cardiac contractility in the animal of step (b) compared tocardiac contractility in the animal of step (a), whereby the detectionof an increase in cardiac contractility identifies a compound that cantreat structural heart disease.
 70. The method of claim 69, wherein thestructural heart disease can be at least one of dilated cardiomyopathyor myocardial contractile dysfunction.
 71. A method of identifying acompound that can treat structural heart disease, comprising: a)measuring brain natriuretic peptide in an animal of claim 51; b)administering the compound to the animal of claim 51; c) measuring brainnatriuretic peptide in the animal of step (b); and d) detecting anincrease in brain natriuretic peptide in the animal of step (b) comparedto brain natriuretic peptide in the animal of step (a), whereby thedetection of an increase in brain natriuretic peptide identifies acompound that can treat structural heart disease.
 72. The method ofclaim 71, wherein the structural heart disease can be at least one ofdilated cardiomyopathy or myocardial contractile dysfunction.
 73. Amethod of identifying a compound that can treat structural heartdisease, comprising: a. measuring brain natriuretic peptide in an animalof claim 58; b. administering the compound to the animal of claim 58; c.measuring brain natriuretic peptide in the animal of step (b); and d.detecting an increase in brain natriuretic peptide in the animal of step(b) compared to brain natriuretic peptide in the animal of step (a),whereby the detection of an increase in brain natriuretic peptideidentifies a compound that can treat structural heart disease.
 74. Themethod of claim 73, wherein the structural heart disease can be at leastone of dilated cardiomyopathy or myocardial contractile dysfunction.